The skin as an organ is of interest from biological, medical, and cosmetological points of view. There are a large number of skin diseases that are either organ-specific, e.g. psoriasis and eczemas, or are manifestations of general disease, such as general allergic reactions. The fact that there are skin-specific diseases can be considered as a proof of the existence of molecular mechanisms that are unique for the skin. Analogously, studies on skin-specific molecular processes are of importance for the understanding and treatment of skin disorders. It seems reasonable to assume that several of these processes in one way or another are related to the most specialized function of the skin, that is the formation of a physico-chemical barrier between body exterior and interior. The physico-chemical skin barrier is localized in the outermost layer of the skin, the stratum corneum.
The stratum corneum is the most specialized structure of the skin. It is the end product of the differentiation process of the epidermis, that is the stratified squamous epithelium that accounts for the outermost portion of the skin. The majority of the cells of the epidermis consist of keratinocytes in various states of differentiation. The lowermost keratinocytes, the basal cells, reside on a basal membrane in contact with the dermis, that is the connective tissue of the skin, and are the only keratinocytes that have dividing capability. A fraction of the basal cells continuously leaves the basal membrane and goes through a differentiation process, which eventually makes the cells become building blocks of the stratum corneum. In this process the keratinocytes go through a number of adaptive changes. There is an increased content of cytoskeleton consisting of epidermis-specific cytokeratins. The intermediate filaments of contiguous cells are joined to a functional unit by an increased number of desmosomes. The most dramatic changes take place during the transition from the uppermost living cell layer, the stratum granulosum, to the non-viable stratum corneum in a process usually called keratinization. Covalently cross-linked proteins are deposited close to the inner aspect of the plasma membrane, forming a very resistant cell envelope. Furthermore a lipid-rich substance, originating in a keratinocyte-specific cell organelle, is secreted to the extracellular space and, by forming lipid lamellae, which surround the cells of the stratum corneum, constitutes the permeability barrier to hydrophilic substances. Finally all intracellular structures except the densely packed cytokeratin filaments disappear.
The cells of the stratum corneum, the corneocytes, are thus non-viable. This means that the regulation of various processes in the stratum corneum must be the result of a “programming” at a state where the keratinocytes are still viable. The turnover of the epidermis, which normally proceeds in about four weeks during which the cells are part of the stratum corneum for about two weeks, is ended by means of cell shedding from the skin surface in the process of desquamation. This process is an example of “programming” of the stratum corneum. A prerequisite for the function of the stratum corneum as a physico-chemical barrier is that its individual cells are held together by mechanically resistant structures, that is desmosomes. The degradation of desmosomes, which is a prerequisite for desquamation, must be regulated so as to give a cell shedding from the skin surface which balances de novo production of the stratum corneum without interfering with the barrier functions of the tissue.
Disorders of Keratinization
Under a large number of pathological conditions in the skin of varying severity, there are disturbances in the keratinization process. In psoriasis there is, in addition to a typical chronic inflammation, overproduction of an immature stratum corneum resulting in the typical scaling of this disease. There is a group of inherited skin diseases characterized by a thickened stratum corneum which leads to the formation of “fish scales”, the so-called ichthyoses. In several of the ichthyoses there is a decreased rate of desquamation. Although less severe than the ichthyoses, “dry skin” (xeroderma) is also characterized by a stratum corneum from which corneocytes are shed, not as under normal conditions as single cells or as small aggregates of cells, but as large, macroscopically visible scales. This disorder is very common among elderly people and among atopics, that is individuals with a decreased resistance to skin irritants and a disposition to develop a characteristic form of endogenous eczema. In the acne diseases there is a disturbed keratinization in the ducts of the sebaceous glands, which leads to the formation of comedones and plugging. The formation of comedones precedes and is believed to provoke the inflammatory acne lesion.
Proteolytic Enzymes are Involved in Keratinzation
There are several stages in the keratinization process and during the turnover of the stratum corneum where proteolytic enzymes seem to play important roles. Certainly the disappearance of all intracellular structures except for the cytokeratin filaments occurring during the transition between viable and cornified epidermal layers must involve proteolysis. The transformation of profilaggrin to filaggrin, a protein that is believed to function in the special type of aggregation of cytokeratin filaments during keratinization, may be catalyzed by a specific proteinase. In the stratum corneum filaggrin is further degraded to low-molecular weight components which are probably important as “natural moisturizers”. Furthermore there are proteolytic modifications of cytokeratin polypeptides during the keratinization process. Finally, proteolytic events are likely to play crucial roles in the degradation of intercellular cohesive structures in the stratum corneum in processes eventually leading to desquamation.
Stratum Corneum Cell Cohesion and Desquamation, the Role of Desmosomes
Intercellular cohesion in the stratum corneum as well as in the viable parts of the epidermis is mediated to a significant extent by desmosomes. A desmosome consists of two symmetrical halves, each of which is formed by two contiguous cells. Each desmosomal half has one intracellular part linked to the cytokeratin filaments and one part made up by glycoproteins anchored intracellulariy and with transmembranal and extracellular parts. The extracellular parts of these proteins, the desmogleins, are adhesion molecules, and through their interaction with each other in the extracellular space a cohesive structure is formed. The degradation of desmosomes seems to follow somewhat different routes in the stratum corneum of palms and soles as compared to non-palmo-plantar stratum corneum. In the latter tissue around 85% of the desmosomes disappear soon after the cells have become fully cornified. The remaining desmosomes, which are preferentially located at the villous edges of the extremely flattened celis, apparently remain intact up to the level where desquamation takes place. In palmo-plantar stratum corneum the corneocytes are much less flattened, and there is no extensive degradation of desmosomes in deeper layers of the tissue, in both tissues desquamation is associated with desmosomal degradation. In ichthyotic skin as well as in “dry skin”, the number of desmosomes in the superficial layers of the stratum corneum has been shown to be increased.
Many of the tissue-specific molecular mechanisms of the skin are associated with the formation and turnover of the barrier-forming outermost layer of the epidermis, the stratum corneum, consisting of cornified epithelial cells surrounded by highly organized lipids. The stratum corneum is continuously being formed in the process of epidermal differentiation. In the efforts to understand the mechanisms by which a constant thickness of the stratum corneum is maintained via a continuos desquamation of surface cells, two human serine proteases, stratum corneum chymotryptic enzyme (SCCE) and stratum corneum tryptic enzyme (SCTE) have been identified (Hansson et al. 1994 and Brattsand et al. 1999). The cloning and expression of SCCE is described in WO95/00651, which hereby is incorporated by reference. Both enzymes belong to the kallikrein group of serine proteases, the genes of which are localized to a short stretch at chromosome 19q13.3-19q13.4 (Diamandis et al. 2000). SCCE is synonymous with human kallikrein 7 (KLK7). It should be noted however, that the numbering of kallikreins is not consistent between species. The expression of SCCE and SCTE seems to be restricted to squamous epithelia undergoing cornification and in which there is a need for desquamation (Ekholm et al. 2000).
Common inflammatory skin diseases may result in severe handicap by causing reduced function, stigmatization, and almost unbearable sensory symptoms. A dominating symptom of many of these diseases is itch, which in many instances may be extremely troublesome, causing severe disturbances in many aspects of every day life and sleeping patterns of sufferers. In atopic dermatitis, affecting more than 10% of children at some point of their childhood, pruritus is a major diagnostic criterion and always present in active disease. It has even been stated that “atopic dermatitis is an itch that when scratched erupts”, and that “pruritus must be considered a quintessential feature of atopic dermatitis” (Beltrani, 1999). The mechanisms of itch are poorly understood, and available treatments are often unsatisfactory. This may be due, at least in part, to lack of satisfactory animal models (Greaves and Wall, 1996).
In inflammatory skin diseases such as psoriasis and atopic dermatitis evidence in favor of a central role for the immune system in pathogenesis is overwhelming. It seems likely that the development of the various disease-specific skin lesions and signs is the result of interactions at the cellular and molecular level between the immune system and skin-derived structures and molecules. In most studies aimed at understanding these interactions focus has been on cytokines, growth factors, and adhesion molecules. Although many of these components are produced by skin cells, they are not unique for the skin, but are more or less generally present in cells and tissues throughout the body. This fact may cause problems in e.g. development of skin-specific therapies. The situation would be different if one could find a truly skin-specific structure or molecule with a central role in the pathophysiology of inflammatory skin diseases. The present invention present new evidence that the serine protease stratum corneum chymotryptic enzyme (SCCE) may belong to this category of skin-specific molecules.